Actinobacillus actinomycetemcomitans (Aa) is an important pathogen in the human oral cavity associated with localized juvenile periodontitis. The bacterium expresses several factors that may contribute to its virulence, one of which is a leukotoxin that kills human white blood cells. This might impair the immune response against Aa. Our studies suggest that the toxicity of Aa dramatically increased when a small DNA sequence was deleted from the promoter sequence of leukotoxin (ltx) operon or when an insertion element is present upstream of the operon. Both events result in a 10-fold increase in leukocyte expression. In the present application, we propose to determine how these events influence the overall expression of the ltx operon in Aa. This will be accomplished by evaluating the specific sequences that control ltx expression and determining if two promoters are functional in highly toxic Aa strains. We will also determine if the deletion removes a sequence that normally functions to repress ltx expression in Aa. Loss of repression may contribute to the constitutively high ltx expression found in highly toxic Aa strains. We will also investigate the mechanism whereby the acquisition of a transposable element stimulate ltx expression. We will determine if a new fusion promoter forms upon insertion of the IS element and/or if the IS element disrupts an existing regulatory sequence that controls ltx expression. Finally, we will determine if toxin-containing vesicles can function to deliver virulence factors (i.e., leukotoxin) to the target cell if concentrated form and if the elaboration of these vesicles is dependent upon the levels of leukotoxin expression. These studies will clarify the mechanism lead to the over-expression of leukotoxin in some strains of Aa and may identify potential new targets for anti-microbial therapy. This is clearly important in light of the increasing incidence of multiple drug resistant bacterial pathogens.